NleE substrate recognition 1 Identification of a distinct substrate binding domain in the bacterial cysteine methyltransferase effectors

The type III secretion system (T3SS) effector protein NleE from enteropathogenic E. coli (EPEC) plays a key role in the inhibition of NF-κB activation during infection. NleE inactivates the ubiquitin-chain binding activity of host proteins TAB2 and TAB3 by modifying the NZF domain through S-adenosyl methionine (SAM) dependent cysteine methylation. Using yeast two hybrid protein interaction studies, we found that a conserved region between amino acids 34 to 52 of NleE, in particular the motif 49GITR52, was critical for TAB2 and TAB3 binding. NleE mutants lacking 49GITR52 were unable to methylate TAB3 and wild-type NleE but not NleE49AAAA52, where each of GITR was replaced with alanine, restored the ability of an nleE mutant to inhibit IL8 production during infection. Another NleE target, ZRANB3, also associated with NleE through the 49GITR52 motif. Ectopic expression of an Nterminal fragment of NleE (NleE34-52) in HeLa cells showed competitive inhibition of wild-type NleE in the suppression of IL-8 secretion during EPEC infection. Similar results were observed for the NleE homolog OspZ from Shigella flexneri 6, which also bound TAB3 through the 49GITR52 motif and decreased IL-8 transcription through modification of TAB3. In summary, we have identified a unique substrate-binding motif in NleE and OspZ, which is required for the ability to inhibit the host inflammatory response. _________________________________________ Many bacterial pathogens including enteropathogenic E.coli (EPEC), enterohemorrhagic E. coli (EHEC) and Shigella utilise a type III secretion system (T3SS) to deliver multiple virulence proteins directly into host cells that subvert a diverse range of normal cellular functions (1). During infection, EPEC and EHEC remain extracellular and attach intimately to the apical surface of enterocytes forming attaching and effacing (A/E) lesions. A/E lesions are characterised by localised microvillus effacement and the accumulation of host cytoskeletal proteins beneath the adherent bacteria (2). A/E lesion formation requires the EPEC T3SS and secreted effector proteins, which are encoded on a genomic pathogenicity island called the locus of enterocyte effacement (LEE PAI). EPEC also delivers a repertoire of non-LEE encoded (Nle) effector proteins into host cells, some of which dampen the inflammatory response during infection and allow EPEC to evade early detection by the host immune system (3-10). http://www.jbc.org/cgi/doi/10.1074/jbc.M116.734079 The latest version is at JBC Papers in Press. Published on July 21, 2016 as Manuscript M116.734079 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Sptem er 4, 2016 hp://w w w .jb.org/ D ow nladed from NleE substrate recognition 2 Recently, several T3SS effectors have been described as novel enzymes that target innate immune signalling pathways for the benefit of bacterial survival and dissemination. For example, the EPEC effector NleB1 is a novel glycosyltransferase that modifies death domain proteins with a single GlcNAc residue, and inhibits death receptor-mediated apoptosis thereby promoting the survival of enterocytes during EPEC infection (11,12). The EPEC effector NleC is a zinc metalloprotease that directly cleaves NF-κB Rel proteins, including p65 and p50, as well as p300 (3,4,7,10,13,14). OspF is a T3SS effector of Shigella classified as a dual-specificity phosphatase (DSP) that represses the expression of pro-inflammatory cytokine genes by dephosphorylating activated MAP kinases and thereby blocking histone 3 phosphorylation at serine position 10 (H3pS10) (15). Another EPEC effector, NleE, is a novel Sadenosyl-L-methionine (SAM)-dependent methyltransferase that modifies a cysteine residue in the Npl4 zinc finger (NZF) domain of the host signalling adaptor proteins TAB2 and TAB3 (TAK1-binding proteins 2 and 3) (16). NleE is conserved across all A/E pathogens and has homologue in Shigella spp., termed OspZ (17,18). TAB2 and TAB3 are redundant proteins that are essential for signalling via the Toll-like, IL-1 and TNF receptors. Upon activation, the NZF domains of TAB2/3 bind K63-linked polyubiquitin chains on target proteins, such as the receptor associated ubiquitin ligases TRAF6 and TRAF2. Polyubiquitin chain binding by TAB2/3 allows TAK1 to form a complex with IKK and subsequently phosphorylate IKKβ. This leads to the phosphorylation and degradation of IκB and subsequent activation of NF-κB. NleE abolishes the ubiquitin-chain binding capacity of the NZF domains of TAB2/3 and thereby disrupts NF-κB signalling (16). More recently, the NZF domain containing protein ZRANB3 was identified as an NleE substrate, and when methylated, the NZF domain of ZRANB3 also lost the ability to bind polyubiquitin chains (19). ZRANB3 has been previously described as a translocase or annealing helicase that interacts with K63-linked polyubiquitinated PCNA (Proliferating Cell Nuclear Antigen) (19). Despite the comprehensive work that has defined the novel enzymatic activities of NleE and other T3SS effectors, knowledge of effector-target recognition and binding regions are often lacking. Here, we identified a highly conserved motif in the N-terminals of NleE that is critical for the recognition of targets in host. We also investigated the propensity of OspZ to bind and methylate NleE host targets as well as the role of OspZ in the inhibition of NF-κB activation and IL8 expression during S. flexneri 6 infection. RESULTS Identification of a TAB3 binding domain in NleE. Although the 208IDSYMK214 motif is essential for NleE activity (6), the contribution of this motif to enzyme function is unknown. To test if the 208IDSYMK214 motif was involved in target recognition, we used the yeast two-hybrid system (Y2HS) to investigate binding of NleE to its host target, TAB3. Saccharomyces cerevisiae AH109 was co-transformed with NleE and TAB3 yeast expression constructs and protein-protein interactions were assessed by growth of the yeast on selective quadruple drop-out media (QDO). Full length NleE from EPEC E2348/69 interacted with TAB3 as did NleE6A, a derivative of NleE where each amino acid in the 208IDSYMK214 motif was substituted with alanine (Fig. 1A). Hence despite the fact that NleE6A is unable to inhibit NF-κB activation (6), this mutant derivative still bound to TAB3, suggesting the existence of a substrate recognition domain distinct from the 208IDSYMK214 motif. To narrow down the TAB3-binding region within NleE, we performed a sequential deletion analysis of NleE and determined the capacity of the NleE truncations to bind TAB3 in the Y2HS (Fig. 1A and B). The results suggested that the Nterminal 53 amino acids of NleE were crucial for the interaction with TAB3 (Fig. 1A and B). Within this 53 amino acid sequence, amino acids 34 to 52 are highly conserved among NleE/OspZ proteins from EPEC, EHEC, C. rodentium and Shigella (Fig. 2A). Upon transformation of S. cerevisiae AH109 with constructs expressing TAB3 and just the fragment comprising amino acids 34-52 (NleE34-52) growth was observed on QDO suggesting amino acids 34-52 within NleE were sufficient to sustain an interaction with TAB3 (Fig. 1A and B). To further confirm the interaction, purified GST-NleE and GST-NleE34-52 were used to pull down TAB3-Flag from cell lysates of by gest on Sptem er 4, 2016 hp://w w w .jb.org/ D ow nladed from NleE substrate recognition 3 transfected HEK293T cells (Fig. 2B). GST alone showed no interaction with TAB3-Flag. Contribution of the NleE34-52 TAB3 binding region to the inhibition of NF-κB activation. Based on the observation that the region between amino acids 34 to 52 of NleE was sufficient for binding to TAB3, we tested whether this region was required for inhibition of NF-κB activation by NleE. Using a dual-luciferase reporter system to measure NFκB activation (6), we found that NleE lacking the N-terminal 53 amino acids (NleEΔ1-53), or the region between amino acid 34 to 52 (NleEΔ34-52) was unable to inhibit NF-κB activation (Fig. 2C). HeLa cells expressing EGFP alone were used as a positive control for activation. This suggested that the region between amino acids 34-52 was essential for the function of NleE. We predicted that amino acids 34-52 would overlap the T3SS secretion and translocation signal making analysis of this region of NleE difficult in EPEC. To test this, we employed a system that utilizes translational fusions to TEM1 β-lactamase (20). Equivalent expression of the TEM1-fused NleE derivatives in EPEC E2348/69 was detected with β-lactamase antibodies (data not shown). Not surprisingly, a derivative of NleE lacking amino acids 34-52 was not translocated into host cells (Fig. 3A). To narrow down the TAB3 binding domain of NleE further, we screened three NleE deletion mutants, NleEΔ41-52, NleEΔ45-52 and NleEΔ49-52 for their ability to bind TAB3. None of the NleE mutants tested bound to TAB3 in the Y2HS suggesting that the region between amino acids 49 and 52 was important for NleE-TAB3 interactions (Fig. 3B). Co-immunoprecipitation experiments in cultured epithelial cells confirmed this observation, as full length GFP-NleE immunoprecipitated with TAB3Flag, whereas no binding was observed between TAB3-Flag and a derivative of NleE where each of 49GITR52 was replaced with alanine (GFPNleE49AAAA52) (Fig. 3C). Despite equivalent expression (data not shown), none of the NleE deletion mutants (NleEΔ41-52, NleEΔ45-52 and NleEΔ49-52) were able to inhibit NF-κB activation as efficiently as full length NleE (Fig. 3D), further suggesting that the 49GITR52 region was important for NleE function. HeLa cells expressing EGFP alone were used as a positive control for activation. We then tested the ability of EPEC to translocate NleEΔ49-52, and observed that NleEΔ49-52-TEM1 was translocated into cells (Fig. 3A), making analysis of the 49GITR52 region possible in the EPEC background. Contribution of the NleE 49GITR52 motif to TAB3 cysteine me